Photoactivated antiviral and antitumor compositions

ABSTRACT

Disclosed herein are compounds, compositions, and methods to inactivate a virus and destroy tumor cells. The methods involve the addition into the cell of a compound containing a photosensitizing chemical and an energy donating chemical, optionally linked by a chemical tether. Also introduced into the cell are means to chemically activate the energy donating chemical which photoactivates the photosensitizing chemical which then destroys the tumor or virus. The photosensitizing chemical is preferably hypericin, porphyrin, or an analog and the energy donating chemical is preferably luciferin or an analog. Methods for synthesizing the chemicals are also disclosed. Further, the energy donating chemical is activated by an activating chemical. The expression of the activating chemical is regulated so as to target the virus-infected or tumor cells. Regulating the activating chemical is accomplished by a number of methods including construction of an expression plasmid containing a gene encoding the activating chemical under control of a promoter which is transactivated by replication of the virus or transactivated by elevated levels of proteins expressed in tumor cells.

The following is a continuation-in-part of United States patentapplication Ser. No. 07/995,877 filed Dec. 23, 1992, now abandoned.

FIELD OF THE INVENTION

This invention relates to compositions containing photodynamic moleculesand means for activating the antiviral and antitumor properties of suchmolecules within viral-infected and tumor cells without an externallight source.

BACKGROUND OF THE INVENTION

In recent years, photodynamic therapy (PDT) has emerged as a promisingtool in both antiviral and cancer chemotherapy. In the presence of lightof the appropriate wavelengths, the photoactive molecule absorbs thelight and inactivates the virus or destroys the tumor cell. Photoactivemolecules which are currently employed include a mixture of compoundscalled hematoporphyrin derivatives (HpD), the purpurins and thephthalocyanines. A major drawback is that PDT cannot be extended totreatment in regions of the body where light does not penetrate.

Moan et al., "Yearly Review: Porphyrin Photosensitization andPhototherapy," Photochemistry and Photobiology: Vol. 43, No. 6, pp.681-690 (1986), which is incorporated herein in its entirety byreference, discloses the tumor localizing property of porphyrins andtheir use in PDT. The method of treatment involves direct injection ofthe photosensitizer. The photosensitizer molecules are affected by alight source located outside the body. Moan et al. specifically mentionthat because penetration of light is limited, PDT can never be used toeliminate large tumors.

Meruelo et al., "Therapeutic Agents With Dramatic AntiretroviralActivity and Little Toxicity at Effective Doses: Aromatic PolycyclicDiones Hypericin and Pseudohypericin," Proc. Natl. Acad. Sci., Vol. 85,pp. 5230-5234 (1988), which is incorporated herein in its entirety byreference, have demonstrated that hypericin inhibits the replication ofFriend leukemia virus and radiation leukemia virus, both in vitro and invivo. Meruelo et al. stated that when hypericin was administered to miceat doses sufficient to prevent retroviral-induced disease, the miceappeared to be devoid of undesirable side effects. Meruelo et al. havealso reported that hypericin can reduce the spread of HIV. Meruelo etal. speculate that hypericin may act by direct inactivation of thevirions.

Chanh et al., "Photodynamic Inactivation of Simian ImmunodeficiencyVirus," J. of Virological Methods, Vol. 26, pp. 125-132 (1989), which isincorporated herein in its entirety by reference, disclose photodynamicinactivation of simian immunodeficiency virus (SIV). A dihematoporphyrinether (DHE) was used to inactivate, in vitro, the infectivity of SIV.DHE was activated through the use of a laser beam. The experiment wasconducted by incubating SIV suspended in a culture medium with DHE inthe dark, followed by irradiation. The authors postulated that thistreatment will reduce the risk of infection by enveloped viruses duringtransfusions.

Lavie et al., "Studies of Mechanisms of Action of the AntiretroviralAgents Hypericin and Pseudohypericin," Proc. Natl. Acad. Sci., Vol. 86,pp. 5963-5967 (1989), which is incorporated herein in its entirety byreference, disclose that hypericin and pseudohypericin possessanti-retroviral activity. Specifically, the hypericin andpseudohypericin suppress the spread of murine retrovirus in vitro and invivo. Treatment by hypericin and pseudohypericin resulted in completeinactivation of reverse transcriptase of both murine and human viruseswhen the compounds were administered by injection.

Hudson et al., "Antiviral Activities of Hypericin," Antiviral Research,Vol. 15, pp. 101-112 (1991), which is incorporated herein in itsentirety by reference, disclose that hypericin inactivates murinecytomegalovirus (MCMV) Sindbis Virus, and HIV-1.

U.S. Pat. Nos. 4,898,891, 5,049,589 and 5,047,435 to Lavie et al., allof which are incorporated herein by reference, disclose the antiviraleffects of hypericin and pseudohypericin and antiviral pharmaceuticalcomposition containing hypericin as an active ingredient.

Finally, Matthews et al., "Photodynamic Therapy of Viral Contaminantswith Potential for Blood Banking Applications," Transfusion, Vol. 28,No. 1, pp. 81-83 (1988), which is incorporated herein in its entirety byreference, disclose PDT for eradicating viral contaminants. The methodemploys a hematoporphyrin derivative used as the photosensitizer toinactivate an enveloped virus. The method involves extracorporealplasmaphoresis, i.e., the blood is taken out of the body prior to beingtreated with hematoporphyrin and light. The method uses visible light.

There is a need to connect an energy source to photoactive molecules sothat PDT can be expanded to all regions of the body. There is also aneed to provide a method for targeting PDT to viral infected cells or totumor cells.

Thus, activation of the energy source must be regulated such thatactivation preferentially occurs in the virus-infected cells or tumorcells. The present invention overcomes the problems in the prior art byemploying an energy source which (1) emits energy in a broad band ofwavelengths in the range in which the photoactive molecule absorbs and(2) is chemically activated by another chemical, which is regulated toexpress in the virus-infected or tumor cell. Thus, photoactivation ofthe photosensitizer is targeted to the virus-infected or tumor cells.The inventors have also developed chemical tethers to connect the energysource and the photoactive molecule. The use of such a tethered compoundallows for the in vivo introduction of an internal chemically-activatedlight source having broad applications in antiviral and tumor therapy.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide antiviralcompositions.

It is a further object of the invention to provide tethered compoundsfor use in the antiviral compositions.

Another object of the invention is to provide antitumor compositions.

Another object of the invention is to provide tethered compounds for usein the antitumor compositions.

Another object of the invention is to provide hypericin analogs for useas intermediates in the preparation of the tethered compounds.

Another object of the invention is to provide luciferin and its analogsfor use as intermediates in the preparation of the tethered compounds.

Yet another object of the invention is to provide methods forsynthesizing precursors of the luciferin analogs.

Yet another object of the invention is to provide methods forsynthesizing the tethered compounds.

A still further object of the invention is to provide means foractivating the tethered compounds.

A still further object of the invention is to provide expressionplasmids for activating the tethered compounds.

A still further object of the invention is to provide a liposomecontaining an expression plasmid.

A still further object of the invention is to provide a transfected cellcontaining an expression plasmid.

A still further object of the invention is to provide an eukaryotic cellcontaining a stably integrated copy of an expression plasmid.

A still further object of the invention is to provide a viral vectorproduced from the eukaryotic cell.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by the practice of the invention. Theobjects and advantages of the invention will be attained by means of theinstrumentalities and combinations particularly pointed out in theappended claims.

To achieve the objects in accordance with the purpose of the inventionas embodied and broadly described herein, the present invention providesa composition for inactivating a virus or destroying a tumor cell. Thecomposition preferably contains three components. The first component isa chemical capable of photosensitization, hereinafter termed "aphotosensitizing chemical." The second component is an energy donatingchemical. The third component contains means for activating the transferof energy or the emission of light from the energy donating chemical.The present invention preferably employs luciferin, a natural lightsource, or its analogs as the energy donating chemical. Thephotosensitizing chemical is preferably hypericin, a porphyrin, or oneof their analogs.

In a preferred antiviral composition, as well as in a preferredantitumor composition, the hypericin, porphyrin, or analog is connectedto the luciferin or analog, by way of a chemical tether or chemicallinker. Thus, in a preferred embodiment of the invention, the first andsecond components of the antiviral or antitumor composition, i.e., thephotosensitizing chemical and the energy emitting chemical, areconnected by a chemical tether and form a tethered compound. As usedherein, "chemical tether" or "chemical linker" is a chemical connectorof two ring compounds.

The hypericin-luciferin tethered compound is preferably prepared througha condensation reaction of an activated hypericin with a luciferinanalog. The luciferin analogs are synthesized from known compounds.

The compositions of the present invention also preferably contain athird component that contains chemical means for activating the transferof energy or emission of light from the energy donating chemical. Thechemical means of the present invention is an activating chemicalencoded by a gene under control of regulatory genetic elements. The DNAcontrolling the activating chemical operably contains regulatory motifsrecognized by host cell transcription factors in addition to motifsrecognized by viral regulatory proteins. As used herein, "regulatoryelement" ("regulatory nucleic acid sequence") is a region (sequence)that determines when, if, and at what level the DNA encoding theactivating chemical is expressed. Such regulatory elements includepromoters, enhancers, and transcription and translation initiation andtermination sequences. As used herein, "nucleic acid sequence" refersgenerally to a polynucleotide molecule, more specifically to a linearseries of nucleotides connected one to the other by phosphodiester bondsbetween the 3' and 5' carbons of the two adjacent pentoses.

Luciferin, a preferred energy donating molecule of the presentinvention, transfers energy or emits light when it reacts with theenzyme luciferase, ATP, and molecular oxygen, i.e., when the luciferaseactivates luciferin. Therefore, luciferase is an activating chemicalwhen luciferin is the energy donating chemical. Thus, another preferredembodiment of the invention is regulating the expression of luciferasesuch that luciferase is preferentially or only expressed invirus-infected or tumor cells.

Regulating the activating chemical of an antiviral composition ispreferably accomplished by first constructing an expression plasmidcontaining a gene encoding the activating chemical under control of apromoter. The DNA encoding the activating chemical is inserted into avector, such as an expression plasmid, in proper orientation and correctreading frame for expression. Preferably, the antiviral composition isused to inactivate an enveloped virus but may be used for inactivatingany viruses. More preferably, the antiviral composition is used toinactivate DNA enveloped viruses such as Herpes Simplex Virus and RNAenveloped viruses such as lentiviruses HIV and EIAV. The expressionplasmid preferably contains a luciferase gene under control of anenveloped virus promoter, e.g., the retrovirus long terminal repeat(LTR). The promoter is selected such that replication of the virus willtransactivate the promoter resulting in increased expression ofluciferase leading to activation of luciferin and photoactivation ofhypericin. Thus, photoactivation is localized to the virus-infectedcells, thereby targeting the antiviral activity of the photosensitizingchemical.

The expression plasmid may be introduced into a cell by a variety ofknown methods including incorporation into a liposome. Other knownmethods that may be employed include, but are not limited to, the use ofnaked DNA transfer, microinjection, or calcium phosphate precipitation.A viral vector may also be constructed and introduced into a cell byviral mediated gene therapy or other known gene therapy techniques. Sucha viral vector is preferably constructed by stably integrating a copy ofthe expression plasmid into a cell line. The viral vector is thenproduced from the cell line.

For use in an antitumor composition, the regulation of the activatingchemical is also preferably accomplished by constructing an expressionplasmid. The expression plasmid contains the gene encoding theactivating chemical, e.g., luciferase, in an embodiment when luciferinis the energy donating chemical, under control of a promoter. Thepromoter is transactivated by elevated levels of proteins expressed intumor cells. Transactivating the promoter increases expression ofluciferase which activates luciferin and photoactivates thephotosensitizing chemical, i.e., hypericin, in one embodiment. Thus,photoactivation is localized to the tumor cell, thereby targeting theantitumor activity of the photosensitizing chemical.

The expression plasmid used for regulating the activating chemical ofthe antitumor composition may be introduced into a cell by methodssimilar to those discussed for the antiviral composition. These methodsinclude, but are not limited to, incorporation into a liposome, nakedDNA transfer, microinjection, or calcium phosphate precipitation. Aviral vector may also be constructed and introduced into a cell throughknown gene therapy techniques as discussed above.

The tethered compounds of the present invention are synthesized througha condensation reaction of the energy donating chemical with thephotosensitizing chemical. In preferred embodiments of the presentinvention, the tethered compounds are synthesized by condensing aporphyrin, hypericin or their analogs with luciferin or its analogs.

The antiviral composition of the present invention may be used in amethod for inactivating a virus. In a preferred method, the first andsecond components of the antiviral composition, preferably in the formof a tethered compound, are administered in pharmaceutically effectiveamount to the virus-infected cell. In addition, the third component ofthe antiviral composition, i.e., the component containing the chemicalmeans for activating the transfer of energy or the emission of light, isalso administered to the virus-infected cell, as discussed above, whereit then activates the energy donating chemical which activates thephotosensitizing chemical which inactivates the virus.

The antitumor composition of the present invention may be used in amethod for treating tumors by destroying neoplastic cells. In apreferred method, the first and second components of the antitumorcomposition, preferably in the form of a tethered compound, areadministered in a pharmaceutically effective amount to a neoplasticcell. In addition, the third component of the antitumor composition,i.e., the component containing the chemical means for activating thetransfer of energy or the emission of light, is also administered to theneoplastic cell, as discussed above, where it then activates the energydonating chemical which then activates the photosensitizing chemicalwhich destroy s the neoplastic cell.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graphic representation of the comparison of thechemiluminescent emission spectrum of the luciferase-catalyzed oxidationof luciferin and the absorption spectrum of hypericin in the red regionof the visible spectrum.

FIG. 2 is a graphic representation of the time course of thechemiluminescent emission from the luciferase-catalyzed oxidation ofluciferin. The concentration of the reactants are as follows:luciferase!=2.67×10⁻⁷ M, luciferin!=1.18×10⁻⁶ M, ATP!=5×10⁻⁵ M; thebuffer is 25 mM glycylglycine, 15 mM MgSO₄, 4 mM EGTA, 15 mM K₃ PO₄ atpH=7.75 and the reaction is carried out at 25° C.

FIG. 3A is a representation of an expression plasmid containing theluciferase gene under the control of the EIAV LTR. FIG. 3B is arepresentation of a retroviral vector containing the luciferase geneunder the control of the HIV LTR. This vector may be used for killingHIV/HIV-infected cells. FIG. 3C shows the construct of a retroviralvector for cell-specific killing of melanoma cells.

FIG. 4 shows the antiviral activity of hypericin in the presence ofluciferin and luciferase.

FIG. 5 shows the expression of luciferase in ED cells in the presence ofEIAV or ETat.

FIG. 6 shows 100-1000 fold reduction in virus production in infectedcells treated with the expression plasmid of FIG. 3A and a tetheredhypericin-luciferin compound according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the presently preferredembodiments of the invention, which together with the followingexamples, serve to explain the principles of the invention.

The invention relates to antiviral and antitumor compositions comprisinga first component containing a photosensitizing chemical, a secondcomponent containing an energy donating chemical, and preferably a thirdcomponent containing means for regulating the emission of light ortransfer of energy as well as to methods for synthesizing suchcomponents. The energy donating molecule should emit energy or light ina broad band of wavelengths in the range where the photoactive chemicalabsorbs. As used herein, a photosensitizing chemical is a chemical thatis activated by light or energy transfer.

A preferable natural energy source is luciferin and its analogs. Thereaction of luciferin with the enzyme luciferase, ATP, and molecularoxygen produces an intense long-lived emission from a triplet state,phosphorescence. The light produced by luciferin and its analogs is inthe 520-680 nm region. Luciferin does not emit light or energy unlessactivated. Thus, regulating the expression of luciferase regulates theactivation of luciferin.

The photosensitizing chemical is preferably one selected from hypericin,other quinones, hematoporphyrin derivatives, phthalocyanins andporphyrins. A specific embodiment involves the use of hypericin as thephotosensitizing chemical. Hypericin absorbs light in the 540-660 nmrange. When hypericin is photoactivated, it produces singlet oxygen witha quantum yield of 0.74.

The energy transfer efficiency is optimized by connecting the energydonating chemical to a photosensitizing chemical by way of a chemicaltether. The chemical tether connects the two ring compounds. Thus, apreferred embodiment of the invention includes the first and secondcomponents in the form of a tethered compound.

The tethering of the photosensitizing chemical and the energy donatingchemical is directed to finding the most efficient energy transferbetween the donor and the acceptor. The most efficient transfer dependson the relative separation and orientation of each of the twocomponents.

Specifically, a preferred embodiment is directed to the connectionbetween hypericin and luciferin or its analogs. Hypericin is a preferredphotosensitizing chemical because low dosage administration of hypericinavoids undesirable side effects.

One embodiment of the invention is directed to the use of an activatedhypericin analog, an anhyride or halide, for use as an intermediate inthe preparation of the tethered compound. The anhyride and halide havethe following formula: ##STR1##

The anhyride can be prepared in three steps from hypericin. Acetylationof hypericin followed by chromic acid oxidation generates the diacid,which when reacted with DCC completes the synthesis of the anhydride.The halide is made by acetylation followed by benzylic halogenation.

Another embodiment of the invention is directed to using, as thephotosensitizing chemical, porphyrins of the following formula: ##STR2##wherein Z¹ -Z⁶ are alkyl or alkenyl groups having 1-15 carbon atoms.

Luciferin and its analogs can be used to form tethered compounds withphotoactive chemicals such as phorphyrins, hypericin, or other quinones.The tethered compounds must demonstrate overlap in the absorptionspectra of the selected photoactive chemical with the emissions spectraof the energy donating chemical.

When oxidized, luciferin's broad emission band is centered at 560 nm andoverlaps the two strongly absorbing bands of hypericin in the red regionof the visible spectrum at 555 and 600 nm. See FIG. 1 for a comparisonof the spectrum of the chemiluminescent emission of theluciferase-catalyzed oxidation of luciferin and the absorption spectrumof hypericin in the red region of the visible spectrum. This overlapallows for luciferin to be paired with hypericin.

During the luciferase-catalyzed oxidation of luciferin, one photon oflight is produced per molecule of substrate consumed. FIG. 2 presentsthe time course of the chemiluminesecent reaction of luciferin. Thetethering of luciferin or its analogs must not disrupt the recognitionand binding of the luciferin substrate by the enzyme for the lightproducing reaction, luciferase. Therefore, the preferred luciferinanalogs for use in preparing tethered compounds are of the followingformula: ##STR3## The tether or chemical linker is located at either R₁,R₂, R₃ or R₄. X and X' may be one of the following: S, O, H,H, CH═CH orNR₄. When R₁ is the site of the tether, R₁ is --CO₂ (CH₂)_(n) Y, whereinn is 2-15; Y is OH, SH or NH₂ ; and R₂ -R₄ are H. When R₂ is the site ofthe tether, R₂ can be one of the following: --(CH₂)_(n) CO₂ H and--S(CH₂)_(n) Y, wherein n is 1-15; Y is OH, SH or NH₂ ; and R₁ and R₃-R₄ are H. When R₃ is the site of the tether, R₃ can be one of thefollowing: --CO₂ H and --(CH₂)_(n) Y, wherein n is 1-15; Y is OH, SH orNH₂ ; and R₁ -R₂ and R₄ are H. When R₄ is the site of the tether, R₄ is--(CH₂)_(n) Y, wherein n is 2-15; Y is OH, SH or NH₂ ; and R₁ -R₃ are H.The formula above displays luciferin when R₁ -R₃ are H and both X and X'are both S.

Luciferin analogs may be synthesized in a number of ways, depending onwhich analog is to be used.

First, a benzothiazole intermediate is employed in the formation of aluciferin analog of the above-formula. The benzothiazole employed hasthe following formula: ##STR4## The benzothiazole is synthesized byadding hydrogen sulfide to an electron deficient imino quinone of thefollowing formula: ##STR5## The resulting adduct undergoes dehydrationto form the luciferin analog.

A second intermediate, a benzothiazole hydroxy nitrile has the followingformula: ##STR6## R is Me, H or PhCH₂. The benzothiazole hydroxy nitrileis formed by adding cyanide ion to a chloroalkoxybenzothiazole followedby dealkylation to form the benzothiazole nitrile.

Luciferin analogs are then synthesized by adding cysteine or asubstituted cysteine to either one of the intermediates synthesizedabove.

Once the luciferin analogs are synthesized, these compounds are thenreacted in a condensation reaction with the activated hypericin to formthe tethered molecule of the following formula: ##STR7## X and X' may beone of the following: S, O, H,H, CH═CH or NR₄. The tether or chemicallinker of the tethered molecule occurs in one of four locations on theluciferin analog: R₁, R₂, R₃, or R₄. When the tether is located at R₁,the tethered compound has the following formula: ##STR8## R₁ is --CO₂(CH₂)_(n) Z, wherein n is 2 to 15; and R₂ -R₄ are H; Z is O, S or NH; Ais O or H,H; and G is CH₃, CO₂ H, CO₂ Me, or CH₂ Br.

If R₂ is the site of the tether, then the compound has the followingformula: ##STR9## R₂ may be one of the following: --(CH₂)_(n) COZ and--S(CH₂)_(n+) Z, wherein n is 1-15; R₁, R₃ and R₄ are H; Z is O, NH orS; A is O or H,H; and G is CH₃, CO₂ H, CO₂ Me, CH₂ Br.

When R₃ is the site for the tether, the compound has the followingformula: ##STR10## R₃ may be one of the following: --COZ and --(CH₂)_(n)Z, wherein n is 1-15; R₁ -R₂ and R₄ are H; Z is O, NH, or S; A is O orH,H; and G is CH₃, CO₂ H, CO₂ Me, CH₂ Br.

When R₄ is the site of the tether, the compound has the followingformula: ##STR11## R₄ is --(CH₂)_(n) Z, wherein n is 2-15; R₁ -R₃ are H;Z is O, NH, or S; A is O, or H,H; and G is CH₃, CO₂ H, CO₂ Me, CH₂ Br.

The tethered compound may also contain the luciferin analogs discussedabove linked to the porphyrin compound discussed above. As in theluciferin-hypericin tethered compound, the site of the tether orchemical linker may be at R₁, R₂, R₃ or R₄. The tether is attached tothe porphyrin at either of its two carbonyls. The luciferin-porphyrintethered compounds will be similar in structure to luciferin-hypericintethered compounds except for the substitution of porphyrin forhypericin, i.e., the luciferin and tether or linker portion remain thesame as those described above.

When the tethered compound contains a porphyrin instead of hypericin thecompound has the following general formula: ##STR12## The tether isattached via R₁, R₂, R₃ or X (through NR₄) as explained and definedabove. Z¹ -Z⁶ are each alkyl or alkenyl groups containing 1-15 carbonatoms.

One advantage of combining organic synthesis with virology and molecularbiology is that organic compounds can be designed to exhibit enhancedphysical properties, such as membrane permeability or polarity. As aconsequence, delivery of these compounds can be targeted to subcellularorganelles or to the cytoplasm. The tethered compounds of the inventionare also synthesized by direct, high-yield synthetic routes. Thesesynthetic routes are flexible, permitting the facile generation ofvarious related compounds. Such compounds may be tested initially forphotochemical properties, and promising molecules may then be furthercharacterized biologically. Accordingly, this invention encompasses abroad range of tethered compounds that may be synthesized and tested byknown techniques given the teachings of the present invention.

Particularly preferred tethered compounds are composed of luciferin or aluciferin analog and pseudohypericin (1) or its octahydroxy analog (2),shown below. ##STR13## wherein R₅ is H or CH₃.

Particularly preferred tethered compounds are as follows: ##STR14##wherein R₁ is H, an alkyl or aryl; R₂ and R₃ are H, OH, Cl, CN or analkyl; R₄ is H, OH, an alkyl or aryl, R₅ is H or CH₃ and R₆ is H, OH, Clor an alkyl; X₁ is O, N-alkyl, N-aryl or S and X₂ is O, N-alkyl or S;and n is 1 to 10; and m is 1 to 4. Most preferably, R₁ -R₆ are H and X₁and X₂ are S.

In addition, two general types of tethered compounds may be prepared.One type uses a "caged" luciferin wherein the carboxylic acid grouppresent in luciferin is capped as an activated ester. This ester form ofluciferin will not react with luciferase until it is cleaved byesterases within the cell. This type of tethered compound exhibitsenhanced membrane permeability and is cleaved rapidly by esterasespresent in cells. Preferred tethered molecules of this type are shownbelow (3a-3c). Compound 3c contains four molecules of luciferin permolecule of the hypericin analog. This is significant since once theluciferin interacts with luciferase, it is transformed into a differentcompound which can no longer function as a substrate for luciferase. Thehypericin analog, on the other hand, is a catalyst in this reaction.Alternatively, a compound can be prepared as shown above wherein up to10 or more luciferin molecules are linked together at the CO₂ H and OHends to form a luciferin chain. In such a chain, n is preferably from 1to 10. ##STR15##

Tethered compound 3a may be prepared from commercially available (e.g.,CalBiochem) pseudohypericin (1) and luciferin using isobutylchioroformate as a catalyst. Tethered compound 3b is made usingpseudohypericin, bromoacetic anhydride and luciferin, by first reactingpseudohypericin with bromoacetic anhydride to generate a bromo esterwhich will readily react with the triethylammonium salt of luciferin toform 3b. Tethered compound 3c is prepared from quinone 2 (R═CH₂ OH) andfour molecules of luciferin. Quinone 2 is prepared in three steps from2,6-dichlorobenzoquinone and 2-methyl ethyl acetoacetate. Awater-soluble carbodiimide reagent mediates the reaction between quinone2 and luciferin. A related compound with two methylene groups betweenthe aromatic ring and the luciferin subunit can also be readilyprepared.

A second type of particularly preferred tethered compound may begenerated by combining a "non-caged" analog of luciferin with 1 or 2.These tethered compounds are not cleaved by esterases, and remainchemically linked when added to cells. This enables very efficientintramolecular energy transfer between hypericin and thechemiluminescent intermediate of the reaction of luciferin withluciferase.

We have also developed a particularly preferred enantiospecificsynthesis of luciferin. The major advantage of our synthesis is theconvenient synthesis of luciferin in multigram quantities frominexpensive (less than $1/gram) precursors. Additionally, this routeallows direct access to amino analogs of luciferin. This is significant,since various amino analogs of luciferin, for example the analogdepicted below, react with luciferase. ##STR16## The preferred route forsynthesis of the preferred luciferin analogs (4a-4d) is shown below.Compounds 4a and 4b are prepared from an intermediate, thebenzothiazolehydroxy nitrile second intermediate shown above, in thesynthesis of luciferin. Formation of the allyl ether, Claisenrearrangement and hydroboration are standard organic reactions. Thereaction of the nitrile moiety with cysteine has precedent from theWhite et al., J. Am. Chem. Soc., 83, 2402 (1961), synthesis and from oursynthesis of luciferin. ##STR17## Luciferin analog 4c is prepared inthree steps from an early intermediate in the synthesis of luciferin asshown below. The chloride is converted into a nitrile and a cystein unitis introduced. Alkylation of an amine with bromoethanol is a standardreaction. ##STR18## Luciferin analog 4d is prepared as shown below.Nucleophilic addition to isothiocyanates (R-NCS) followed by alkylationof the resulting intermediate on sulfur with an electrophile is awell-established reaction sequence. ##STR19##

Connection of the luciferin analogs with pseudohypericin, quinone 1,results in the series of particularly preferred "non-caged" tetheredcompounds, 5a-5d, shown below. ##STR20##

There are a number of analogs of luciferin that react with luciferase,including the amino analog and other analogs mentioned above. Theanalogs shown below are also substrates for luciferase. The structuralrequirements for effective substrates are relatively broad, and otheranalogs may be prepared by known techniques given the teachings herein.##STR21##

The tethered compounds of the present invention can contain anycombination of photosensitizing chemicals and energy donating chemicalslinked by a tether, providing that the energy donating moleculeactivates the photosensitizing molecule in sufficient degree to obtainantiviral or antitumor activity. Tether selection is based on (1) therate of transfer from the donor to acceptor and (2) the recognition ofthe substrate by the catalyst for the light producing reaction. The rateand quantum yield of the enzyme catalyzed reaction may be monitored inorder to screen for the potential effects of inhibition of reactivity.

When used for treating virus infections such as DNA or RNA envelopedvirus infections, the first and second components of the antiviralcomposition, preferably in the form of a tethered compound, may beadministered orally, parenterally, and preferably intravenously. In anyevent, a pharmaceutically effective amount of the compound isadministered. An effective amount is determinable by persons skilled inthe art in the view of teachings disclosed herein.

Further, the first and second components, preferably in the form of atethered compound, can be used at dosages containing from about 0.001micrograms to about 100,000 micrograms per kilogram body weight pertreatment, preferably between about 1 microgram and about 5×10⁴micrograms per kilogram of body weight per treatment.

The duration and number of doses or treatments required to control aparticular virus will vary from subject to subject, depending upon theseverity and stage of the illness and the subject's general conditionand will also depend on the activating chemical, as well as the toxicity(if any) of the tethered compound. This will be determinable by personsskilled in the art in view of the teachings contained herein. The totaldose required for each treatment may be administered in divided doses orin a single dose. The preferred form of the first and second components,i.e., the tethered compound, may be administered daily, more than oncedaily, one or two times a week, or as determined by the subject'scondition and the stage of the disease.

Those skilled in the art will appreciate that the frequency of treatmentis subject to optimization, which can be determined by routineexperimentation according to methods well known in the art, e.g., byestablishing a matrix of dosage and frequency and assigning a group ofexperimental subjects to each point of the matrix. Design of thisexperiment will preferably take into account the tissue accumulationproperties of the compounds of the present invention.

The present invention also provides pharmaceutical compositions andformulations for treating lentiviral infections. The first and secondcomponents, preferably in the form of a tethered compound, can beincorporated in conventional, solid and liquid pharmaceuticalformulations (e.g. tablets, capsules, caplets, injectable and orallyadministrable solutions) for use in treating mammals that are afflictedwith viral infections. The pharmaceutical formulations of the inventioncomprise an effective amount of the tethered compounds of the presentinvention as the active ingredients. For example, a parenteraltherapeutic composition may comprise a sterile isotonic saline solutioncontaining between about 0.001 micrograms and about 100,000 microgramsof the tethered compounds of the present invention as described above.It will be appreciated that the unit content of active ingredientscontained in an individual dose of each dosage form need not in itselfconstitute an effective amount since the necessary effective amount canbe reached by administration of a plurality of capsules, tablets,injections or combinations thereof.

Each formulation according to the present invention may additionallycomprise inert constituents, including pharmaceutically-acceptablecarriers, diluents, fillers, salts, and other materials well-known inthe art. Selection will depend upon the dosage form utilized and theparticular purpose to be achieved according to the determination of theordinarily skilled artisan in the field. For example, tablets may beformulated in accordance with conventional procedures employing solidcarriers well known in the art. Examples of solid carriers include,starch, sugar, bentonite, silica and other commonly used carriers.Propylene glycol, benzyl alcohol, isopropanol, ethanol,dimethylsulfoxide (DMSO) dimethylacetamide or other biologicallyacceptable organic solvents or aqueous solutions (e.g. water with a pHhigher than 7 and preferably about 8) may be used as diluents, carriersor solvents in the preparation of solid and liquid pharmaceuticalformulations containing the anti-lentiviral compositions of the presentinvention. Further nonlimiting examples of carriers and diluents includecarbohydrates, albumin and/or other plasma protein components such aslow density lipoproteins, high density lipoproteins and the lipids withwhich these serum proteins are associated. Such lipids includephosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine andneutral lipids such as triglycerides. Additional lipid carriers includewithout limitation tocopherol, retinoic acid and cyclodextranes.Semisolid formulations such as those well-known in the art (e.g.suppositories) are also contemplated.

Preferred parenteral dosage forms may comprise, for example, an isotonicsaline solution, containing between about 0.1 micrograms to about100,000 micrograms of the tethered compounds of the present invention.

Capsules employed in the present invention may be made from anypharmaceutically acceptable material, such as gelatin or cellulosederivatives. Sustained release oral and transdermal delivery systems arealso contemplated, as is interveneous injection.

The antiviral and antitumor compositions containing the preferredluciferin-hypericin tethered molecule requires luciferase for thecatalysis of the light producing reaction and the activation ofhypericin. The activation of the light source is regulated such that thehypericin is photoactivated where needed. The regulation of luciferinactivation is achieved by regulating the expression of luciferase.

The third component of the antiviral composition, i.e., the componentcontaining the chemical means for activating the transfer of energy,regulates the expression of the activating chemical. In a preferredantiviral composition, the activating chemical is luciferase, which isregulated by placing the gene encoding luciferase under control of apromoter that is transactivated by replication of the virus. By placingthe expression of luciferase under control of such a promoter,replication of the virus transactivates the viral promoter resulting inan increased expression of luciferase leading to activation of luciferinand the photoactivation of hypericin. These events are localized invirus-infected cells thereby targeting the antiviral activity ofhypericin.

Specifically, the expression of luciferase is targeted to thevirus-infected cells by constructing an expression plasmid whichcontains the gene coding for luciferase under control of a promoter thatis transactivated by repliction of said virus. Examples of suchpromoters include HIV TAR, the sequence of which is incorporated hereinby reference and which is described in Berkhout et al., Cell, 59:273-282 (1989); Berkhout et al., Cell, 62: 757-767 (1990) and Berkhoutet al., J. Virol., 66: 139-149 (1992), all of which are incorporatedherein by reference, or the consensus enhancer sequence present in thepromoter of Herpes Simplex Virus alpha genes, the sequence of which isincorporated herein by reference, and which is described in Fields etal., ViroloGy, Second Ed. 1990, and Mackem et al. J. Virol, 44: 939-949(1982) each of which is incorporated herein by reference, and the EIAVlong terminal repeat (LTR), the sequence of which is incorporated hereinby reference, and which is shown in FIG. 1 of Carpenter et al., J.Virol., 65(3), 1605-1610 (1991), which is incorporated herein byreference.

FIG. 3A shows a representation of the expression plasmid containing theluciferase gene under control of the EIAV MA-1 LTR. The plasmidcontaining the EIAV LTR is transfected into Cf2th cells and thereexpresses luciferase in the presence of either EIAV or the viraltransactivating protein, Tat, but not in normal Cf2th cells. In oneembodiment, the plasmid is first placed into a liposome before it isdirectly transfected into cells. Similar constructs containing, forexample (FIG. 3B), HIV LTR in place of EIAV LTR express luciferase inthe presence of HIV or HIV Tat. Such HIV constructs can use variousdifferent promotor sequences, provided that at least one or moreTat-responsive cis-acting sequences are included within each promotorsequence controlling luciferase expression. See, e.g., Buchschacher etal., "Human Immunodeficiency Virus Vectors for Inducible Expression ofForeign Genes," J. Virol., 66:5 pp. 2731-2739 (1992) and Buchschacher,"Molecular Targets of Gene Transfer Therapy for HIV Infection," JAMA,269:22, pp. 2880-2886 (1993), each of which is incorporated by referenceherein in its entirety.

Any method of introducing DNA into a cell is sufficient for the genetransfer and therapy herein described. Methods for transferring DNA intocells include, but are not limited to, the use of viral vectors,microinjection, liposome mediated, calcium phosphate precipitation andsimple naked DNA transfer. See Lim et al., Molec. and Cel. Bio., 7(10):3459-3465 (1987); Kasid et al., PNAS, 87: 473-477 (1990); Gilboa,Eli,Retrovirus and Disease, Academic Press, Inc. pp. 95-111 (1989);Kantoff et al, PNAS 83: 6563-6567 (1986); Kasid et al., PNAS, 87:473-477 (1990); Kantoff et al., J. Exp. Med, 166: 219-234 (1987);Cornetta et al., J. Virol. Meth., 23: 187-194 (1989); and Culver et al.,PNAS, 88: 3155-3159 (1991). The disclosure of each of these articles isincorporated herein by reference.

The third component of the antiviral composition, i.e., the componentcontaining the means for activating the transfer of energy, ispreferably introduced into patients through gene therapy techniques. Inone embodiment, the therapy involves retroviral mediated gene therapythat includes construction of a retroviral vector in which the DNAencoding the activating chemical, e.g., luciferase, is placed under thecontrol of a modified retroviral promoter which is activated invirus-infected cells. In one embodiment, the therapy involves firstconstructing a plasmid vector containing a retroviral LTR, retroviralpackaging sequences, and the DNA encoding luciferase. Additionally, thevector may contain DNA encoding a selectable marker, e.g., neomycinresistance.

The plasmid is introduced into a eukaryotic cell or cell line,preferably a packaging cell, which harbors stably integrated proviralsequences sufficient for expression of retroviral structural proteins,but which are deficient in sequences required for packaging andreplication of RNA transcribed by proviral DNA. Following introductionof the plasmid vector, cells containing stably integrated plasmid DNAare identified by expression of the selectable marker. Encapsidation ofvector sequences by the proviral structural proteins results inproduction of retrovirus particles which contain genetic materialencoding the activating chemical under the control of a regulatedpromoter. Transfected cells which produce such retrovirus particles arereferred to as producer cells, and the virus particle produced fromthese cells is referred to as the retrovirus vector.

The vector may be introduced into human and mammalian cells in a varietyof methods. One method comprises removing cells, e.g., lymph cells fromthe patients infected with the virus to be inactivated. The removedcells are then infected with the constructed viral vectors or by othermeans of introducing DNA such as liposome mediated transfer. If the DNAis introduced by liposome mediated transfer, the liposome containsplasmid DNA containing the promoter controlling expression of theactivating chemical, the activating chemical and in some embodiments, aselectable marker. The patient cells containing the gene encoding theactivating chemical are selected through the use of the selectablemarker, i.e., cells are selected if they demonstrate neomycinresistance. The selected cells are then grown and reintroduced into thepatient. Alternatively, the producer cell lines, constructed asdescribed above, are introduced directly into the patient, resulting ininfection in vivo. Further, it is possible to introduce the vectorsdirectly into the patient.

In each of the above-described embodiments, the DNA encoding theactivating chemical is preferably stably integrated within the patient'scell. Expression of the activating chemical is regulated, such that onlyvirus-infected cells express high levels of the activating chemical. Thefrequency of administering the third component via gene therapy or otherknown techniques will depend on how long the inserted DNA can beexpressed and how long the cells containing the inserted DNA willsurvive.

In one specific embodiment of the invention, the tethered compound isused to inactivate HIV, an RNA lentivirus. The tethered compoundcomprises hypericin and a luciferin analog, the first and secondcomponents, and is administered as discussed herein. The third componentis administered by constructing a viral vector containing (1) a promoterthat contains HIV TAR and upstream NF-kB and SP-1 sites, or sequencesnecessary for TAT-mediated transactivation, (2) the packaging sequences,(3) the luciferase gene and (4) Neo^(r), a neomycin resistance marker.The packaging cell line includes a provirus expressing HIV env/gp 120 totarget CD4 positive cells in which HIV replicates, and no packagingsequence. The resultant virus vector line is infected into patient cellseither in vivo or ex vivo. Once the genetic material is integrated intothe patient's cells, luciferase is expressed to high levels only in HIVinfected cells. The expressed luciferase activates the luciferin analogof the administered tethered compound. The luciferin analog in turnactivates the photosensitizing chemical of the administered tetheredcompound. The photosensitizing chemical, once activated by the luciferinanalog, inactivates HIV.

The means for regulating the activating chemical in the preferredembodiment are not the same when the composition is used to destroytumor cells. Specifically, the third component of an antitumorcomposition containing DNA encoding an activating chemical is alsolocated on an expression plasmid. The DNA, however, is under control ofa different promoter, such as the carcino-embryonic-antigen (CEA)promoter. Increased expression of the activating chemical occurs whenlevels of certain proteins, e.g., CEA protein, are elevated. Thus, thepromoter is activated by levels of certain proteins that are elevatedonly in tumor cells. Therefore, the expression of the activatingchemical, i.e., luciferase, is localized to the tumor cells. Otherpromoters useful for targeting photoactivated tumor cell destructionaccording to the invention include, but are not limited to, thetissue-specific tyrosinase promoter (see FIG. 3C and Example 9).

Gene transfer into tumor cells occurs through methods as those describedherein for transfer into virus-infected cells, i.e., via the use ofknown methods such as viral vectors, liposome mediated transfer,microinjection and naked DNA transfer.

The invention also provides a method for destroying noeplastic cells,preferably malignant cells. Thus, it can be used to treat tumors,preferably cancers. The method for destroying a neoplastic cell issimilar to that of inactivating a virus in that the photosensitizingchemical (component one) and the energy donating chemical (componenttwo), preferably in the form of a tethered compound, are introduced intothe tumor cell along with the means for activating the energy donatingchemical (component three). However, in this embodiment, elevated levelsof certain proteins present in tumor cells increases expression of theactivating chemical, which then activates the energy donating chemical,which photoactivates the photosensitizing chemical, resulting indestruction of the tumor cell. Expression of the activating chemical maybe regulated by various means as discussed above.

It is to be understood that the application of the teachings of thepresent invention to a specific problem or environment will be withinthe capabilities of one having ordinary skill in the art in light of theteachings contained herein. Examples of the compounds and compositionsof the present invention and methods of their preparation and use appearin the following examples.

EXAMPLE 1 Preliminary Testing Of An Antiviral Composition ContainingHypericin And Luciferin

Initially, hypericin and luciferin/luciferase (commercially available)were mixed under aerobic conditions in the dark in the presence of EIAVaccording to the following protocol:

Materials and Methods

Serial ten-fold dilutions of EIAV in Hank's balanced salt solution weremixed with an equal volume of luciferase assay buffer containing 25 mMglycylglycine, pH 7.8, 15 mM MgSO₄, 4 mM EGTA, 150 mM KPO₄, 2 mM ATP,and 1 mM DTT. Luciferin and luciferase were added to finalconcentrations of 0.4 mM and 1.6×10⁻⁷ M, respectively. The reactionswere incubated in the dark for 45 min, and ten-fold serial dilutionswere inoculated onto 10⁶ ED cells in the presence of polybrene. Cellswere incubated 5 days at 37° C., fixed in 100% methanol and stained forthe presence of EIAV as previously described (Carpenter et al., J.Virol, 65(3): 1605-1610 (1991), incorporated in its entirety herein byreference). Foci of EIAV-infected cells were quantitated and the resultsexpressed as focus-forming units per ml (FFU/ML).

Results

Hypericin activation by chemiluminescence was dependent on theconcentration of hypericin (FIG. 4). Approximately 90% reduction invirus production was observed with 10 μg/ml hypericin. The antiviralactivity of hypericin was not as efficient as what we previouslyobserved using white light (Carpenter and Kraus, Photochem andPhotobio., 53(2): 169-174 (1991) incorporated in its entirety herein byreference). However, the results demonstrate that theluciferase-catalyzed luciferin reaction photoactivates hypericin invitro.

EXAMPLE 2 Synthesis Of Hypericin Analogs For Use In Preparing TetheredCompounds

Analog A--Hypericin Anhydride

To a stirred suspension of the diacid formed by acetylation of hypericinfollowed by chromic acid oxidation, (H. J. Banks et al. Aust. J. Chem.,1976,29,1509) in ether-chloroform at 0° C. was addeddicyclohexyl-carbodiimide (1.3 eq). The suspension was allowed to slowlywarm to RT over one day. A precipitate was filtered. The filtrate wasconcentrated to give the anhydride (unstable) which was immediatelyreacted with the luciferin analogs synthesized as described in theexamples below.

Analog B--Bisanthraquinone

A solution of bianthraqunoyl (50 mg, 0.088 mmol) in 14 ml of AcOH/CH₂Cl₂ (1:1) was cooled to 0° C. Then a solution of Pb(OAc)₄ (117 mg, 0.26mmol) in 6 ml of AcOH/CH₂ Cl₂ (1:1) was added dropwise at 0° C. Themixture was stirred at 0° C. for 2 hours and was poured into 2N HCl (100ml). Then the mixture was extracted with AcOEt (30 ml×3), washed with H₂O, dried over Na₂ SO₄. After the solvent was removed under reducedpressure, the residue was purified by flash chromatography on silica geleluted with Hexane/AcOEt (2:1), AcOEt/EtOH (9:1) to providebisanthraquinone 39 mg (78%). ##STR22## Analog C--Bianthraqunoyl

A solution of anthrone (500 mg, 1.75 mmol) in 15 ml EtOH was heated toreflux. Then a solution of FeCl₃ (H₂ O)₆ (550 mg, 2 mmol) in 30 ml ofEtOH was added dropwise over 10 minutes and the mixture was stirred atrefluxing for 1 hour. The reaction mixture then was poured into 2% HCl(300 ml) and extracted with AcOEt (50 ml×3). The AcOEt layer was washedwith H₂ O, brine, and dried over Na₂ SO₄. After the solvent was removed,the residue was purified by flash chromatography on silica gel elutedwith Hexane/AcOEt (2:1) to provide bianthraqunoyl 326 mg (81%).##STR23## Analog D--Bisanthraquenone

To a solution of anthrone (40 mg, 0.14 mmol) in pyridine (1 ml),piperidine (90 mg), pyridine-N-oxide (100 mg) and FeSO₄ (5 mg) wereadded. The mixture was heated to 100° C. for 1 hour and poured into 100ml of 3N HCl solution. The mixture was extracted with AcOEt (30 ml×3)and the AcOEt layer was washed with H₂ O, and dried over Na₂ SO₄. Aftersolvent was removed under reduced pressure, the residue was purified byflash chromatography on silica gel eluted with Hexane/AcOEt (2:1),EtOAc, EtOAc/EtOH (7:1). The red fractions were combined to providebisanthraquenone 25 mg (63%). ##STR24##

EXAMPLES 3-4 Synthesis Of Precursors For Luciferin And Its Analogs

Precursor A--Benzothiazole

To a stirred solution of the methyl ester of 2-hydroxy-5-aminobenzoicacid was added ethylchlorooxalate (1.2 eq) in pyridine (1.3 eq) and inmethylene chloride (1M) at 0° C. After stirring overnight, the solventwas removed and the residue was chromatographed using hexanes:ethylacetate.

The diester amide (1 eq) was dissolved in acetic acid (1M). To thissolution was added 1.2 eq of lead tetraacetate. A precipitate whichdeveloped was filtered, washed and taken on to the next step.

The iminoquinone (1 eq) was dissolved in pyridine (1M) and reacted withexcess hydrogen sulfide at 0° C. After warming to RT overnight, thesolution was heated to 50° C., cooled and concentrated in vacuo. Theresidue was purified by chromatography using ethyl acetate:methylenechloride to afford the benzothiazole in 45% yield over two steps. 2: NMR(CDCl₃): 1.17(t, J=7 Hz, 3H), 3.75(s, 3H), 4.25(q, J=7 Hz, 2H), 7.45(ABquartet, 2H).

Precursor B--benzothiazole hydroxy nitrile

To a solution of 1 equivalent of 2-chloro-6-alkoxybenzothiazole (C. G.Stuckwisch J. Am. Chem. Soc., 1949, 3417.) in DMSO (1M) was added 5 eqof sodium cyanide. The solution was heated to 80° C. for 8 h and thenallowed to cool to RT overnight. After workup, the residue was purifiedby column chromatography using hexanes:ethyl acetate to form the cyanobenzothiazole in 60% yield. The compound where R was methyl wasidentical to an authentic sample purchased from Aldrich ChemicalCompany.

The cyano benzothiazole (1 eq) was dissolved in methylene chloride (1Msolution), cooled to 0° C. and treated with 1.5 eq of boron tribromideor excess boron trichloride gas. After allowing the solution to warm toRT overnight, the solvent was removed in vacuo and the residue waspurified by chromatography using hexanes:ethyl acetate to producebenzothiazole hydroxy nitrile in 75% yield. The spectrum of thismaterial was identical to material prepared by reaction with pyridiniumhydrochloride (methyl ether case).

EXAMPLE 5 Synthesis Of Luciferin And Its Analogs From Precursors

Precursors A and B synthesized above were each separately reacted with asubstituted cysteine according to the method of White et al., J. Am.Chem. Soc., 91,2178 (1969), incorporated herein by reference, to formluciferin or a luciferin analog.

EXAMPLES 6A--6D Coupling Of Activated Hypericin And Porphyrin AnalogsWith Luciferin Analogs To Form The Tethered Compounds

6A--From the anhydride of hypericin diacid

To a 0.5M solution of hypericin anhydride (1.0 eq) in 1:1 methylenechloride/DMF at 0° C. is added a solution of the luciferin analog (1.1eq). The solution is allowed to stir to RT over 5 hours. The solventsare removed in vacuo to afford the crude tethered molecule plus a smallamount of luciferin analog. The crude product is partitioned betweencold saturated sodium bicarbonate and ether. The aqueous layer isacidified with cold 1M HCl to afford the tethered molecule as anamorphous solid.

6B--From bis-bromomethyl hypericin

To a 1M solution of luciferin analog (1 eq) in 1:1 methylenechloride/DMF at 0° C. is added sodium hydride (1.0 eq). The suspensionis stirred at 0° C. for 30 min and 1-2M solution of bis-bromomethylhypericin (1 eq) in DMF is added at a rate of approximately 1mmol/second. The reaction is allowed to warm to RT over 5 hours. Thetethered molecule is isolated by concentrating the solution in vacuofollowed by trituration with ether.

6C--From activated porphyrins

To a solution of 1 eq of protoporphyrin IX commercially available fromAldrech Chemical Company in 1:1 methylene chloride/DMF at RT is addeddicyclohexylcarbodiimide (1 eq), followed by the luciferin analog (1eq). The solution is stirred at RT for 8 hours. The solvents are removedin vacuo and the crude product is treated with cold saturated sodiumbicarbonate and filtered to remove dicyclohexylurea. The aqueous layeris carefully acidified with cold 1M HCl. The tethered molecule separatesas an amorphous solid.

6D--From pseudohypericin and "caped" luciferin

To a solution of luciferin (3 mg, 10.7 μmol) and HMM (2.2 mg, 21.8 μmol)in CH₃ CN (1 ml) at -15° C. was added benzyl chloroformate (3.8 mg, 22.3μmol). The mixture was stirred at -15° C. for 15 min. and BtOH (1.5 mg,11 μmol) was added. After 15 min., pseudohypericin (5 mg, 9.6 μmol) inDMF (0.4 ml) was added and the mixture was stirred at RT for 12 hr. Thesolvent was removed under reduced pressure at 23° C. and the darkresidue was purified by chromatography on Sephadex LH-20 eluted withCHCl₃ --MeOH--H₂ O (5:4:1) at 10° C. to provide the ester as a darksolid (3 mg).

EXAMPLE 7 Expression Of Luciferase Under Control Of EIAV Promoter

Luciferase was generated using a luciferase gene located in anexpression plasmid constructed as explained below. The gene was undercontrol of an EIAV long terminal repeat promoter. Plasmid DNA wasintroduced into equine cells in vitro and cell lysates were tested usingfunctional assays of luciferase activity.

Materials and Methods

Plasmids.

A complete proviral clone of the MA-1 isolate of EIAV in lambda EMBL4,designated EIAV 253, Carpenter et al., J. Vir. 65(3): 1605-1610 (1991),incorporated in its entirety herein by reference, obtained according tothe methods disclosed in Carpenter et al. J. Vir., 65(3): 1605-1610(1991), was digested with EcoRl and the proviral insert was ligated toEcoR1 restricted pUC19 using conventional cloning techniques. A plasmidcontaining the complete EIAV provirus, p26A, was identified by colonyblot hybridization and restriction endonuclease digestion. p26A wasdigested with BstN1 and Nar 1 and the 322 base pair (bp) fragmentcontaining the complete MA-1 long terminal repeat (LTR) and 4 bp offlanking sequences was separated by electrophoresis and isolated byelectroelution. See Carpenter et al., J Virol., 65(3): 1605-1610 (1991),FIG. 1, incorporated herein by reference, for the partial sequence ofMA-1, which is also incorporated by reference. The LTR is located atpositions 7909-8231. The ends were filled in with the Klenow fragment ofE. coli DNA polymerase I, modified by the addition of HindIII linkers,ligated to HindIII restricted pUC19, and the fragment was transformedinto E. coli JM109. Colonies containing the LTR insert were identifiedas before and individual plasmids were purified by replating. PlasmidDNA was isolated by ion exchange chromatography and the LTR fragment wasexcised by HindIII digestion and purified by electrophoresis andelectroelution. The LTR fragment was ligated to HindIII restrictedpGL-Basic (Promega Biotec, Madison, Wis.), transformed into JM109, andpositive colonies identified by blot hybridization. Plasmid DNA wasisolated from twelve of the hybridization positive colonies and theorientation of the LTR insert with respect to the luciferase gene wasdetermined by restriction endonuclease digestion. Single clonescontaining the LTR insert in either the forward (pMA-1 LTR/LucF) orreverse (pMA-1LTR/LucR) orientation were selected for further analysisin functional assays of gene expression.

Additionally, a control plasmid having luciferase under control of theSV40 promoter, another plasmid encoding luciferase but no promoter, pGL2Control and pGL2 Basic, were purchased from Promega Biotech (Madison,Wis.) for use in a luciferase assay. The plasmid, pRS Etat M, wasprovided by Dr. David Derse, National Cancer Institute, Frederick, Md.This plasmid expresses the EIAV transactivating protein, Tat, under thecontrol of the Rous sarcoma virus promoter.

Transfection

Cells used for these studies included equine dermal (ED) cells (ATCCCCL57) and ED cells chronically infected with the MA-1 isolate of EIAV,Carpenter et al., J. Virol., 65(3): 1605-1610 (1991) and Carpenter etal. J. Virol., 63: 2492-2496 (1989), both of which are incorporated intheir entirety by reference. For analysis of luciferase expression,cells were seeded in 60 mm tissue culture plates at 5×10⁵ cells/plateand transfected the following day using Transfectase reagent (BRL). Inmost cases, cells were transfected with 10 μg of the luciferaseexpression plasmids (pMA-1 LTR/LucF, pMA-1 LTR/LucR, pGL2 control, orpGL2 basic) in the presence or absence of 100-300 ng pRS Etat-M. At 48hr post-transfection, cells were lysed and assayed for luciferaseexpression using commercially available reagents (Promega). Comparablelysis buffers and assay reagents prepared in the laboratory were alsotested.

Luciferase Assay

Assays for luciferase activity measure the production of light followingthe addition of the substrate, luciferin. Ten μl of cell lysate wasmixed with 350 μl of reaction buffer containing 25 mM glycylglycine, pH7.8, 5 mM ATP, 15 mM MgSO₄, pH 7.8, 4 mM EGTA, and 1 mM DTT. The sampleswere placed in an SLM 8000° C. spectrofluorometer, injected with 200 μlof 0.2 mM luciferin in 25 mM glycylglycine, 15 mM MgSO₄, 4 mM EGTA, 2 mMDTT, and light output was measured at 560 nm for 60 sec.

Results

Plasmids containing the MA-1 LTR upstream of the luciferase gene wereobtained by standard cloning techniques as described above. Plasmidswere functionally characterized in ED cells, in MA-1 infected ED cells,and in ED cells co-transfected with EIAV Tat (ETat). Only baselinelevels of luciferase were expressed in ED cells in the absence of eithervirus infection or virus replication (FIG. 5). Expression of luciferasewas detectable in both virus-infected cells, and in cells co-transfectedwith ETat. The values obtained from these experiments were extrapolatedto a standard curve derived using known molar concentrations ofcommercially available luciferase (Sigma, St. Louis, Mo.). The molarconcentrations of luciferase in the cell lysates ranged between1.8×10⁻¹³ to greater than 3×10⁻¹² M. The relatively low levels ofluciferase were likely due to the low transfection efficiency of EDcells (Carpenter, unpublished observations).

EXAMPLE 8 Assessment Of Antiviral Activity In Cells

As a model system for development of inducible chemiluminescence, weconstructed a further plasmid containing the promoter region of EIAVinserted upstream of the gene for luciferase (pMA-1LTRLuc). Calciumphosphate co-precipitation was used to co-transfect Cf2th cells withpMA-1LTRLuc and pSVNeo5, and cells were selected for neomycin resistanceusing G418. Resistant cells were expanded, analyzed for the presence ofpMA-1LTRLuc using the polymerase chain reacation (PCR). Cells containingthe luciferase gene were transfected with an EIAV Tat expression plasmidand tested for expression of luciferase activity in transient expressionassays. Twelve of 24 cell lines derived from single-cell clones werepositive by both assays. These cells were subcultured, and parallelcultures were infected with the MA-1 isolate of EIAV. Initial assayswere done to determine if incresed levels of luciferase expressionoccurred in virus-infected cells. Results (Table 1) indicated thatluciferase expression increased 10-1000 fold in virus-infected cells, ascompared to non-infected cells, thus demonstrating the targeting ofhypericin activation to infected cells. The matched cell lines 18-4 and18-4/MA-1 provided a useful model to assess the chemiluminescentactivation of hypericin in reducing production of a lentivirus in cellsexpressing luciferase.

                  TABLE 1                                                         ______________________________________                                        Fold-transactivation of luciferase expression by                              EIAV in stable cell lines transfected with pMA-1LTRLuc                               nM LUCIFERASE                                                          CELL CLONE                                                                             Uninfected MA-1 Infected                                                                             FOLD-INCREASE                                 ______________________________________                                        13-2     0.13       1.9         15                                            13-4     0.008       0.95       118                                           14-1     0.14       1.0          7                                            14-2     0.19       1.5          7                                            14-3     0.02       1.1         55                                            17-1     0.007       0.65        9                                            17-4     0.017      1.7         100                                           18-1     0.015       0.66       44                                            18-2     0.023      2.1         91                                            18-3     0.006       0.74       123                                           18-4     0.002      2.0         1,000                                         19-1     0.016      1.5         94                                            ______________________________________                                    

CF2th cells infected with EIAV (Cf2th/EIAV) and EIAV-infected Cf2thcells expressing the luciferase gene (18-4/EIAV) were seeded induplicate in a six-well tissue culture plate. At two days, culturesupernatant was removed and the cells were washed 3× with cell culturemedia. The remainder of the experimental protocols were completed invery subdued light. One well each of Cf2th/EIAV and 18-4/EIAV weretreated with the tether molecule of Example 6D, 60 μg in 3 ml media. Theother wells served as non-treated controls. Cells were incubated at 37°C. for one hour, media was aspirated, and cells were washed 3× and freshmedia was added. At 0, 13, and 44 hours, culture supernatant was sampledfrom each well and virus production was quantitated using a focalimmunoassay. Briefly, equine dermal cells were inoculated with serialten-fold dilutions of supernatant samples, and after five days foci ofinfectious virus was detected by immunocytochemistry.

Results indicated that production of infectious virus was reduced100-1000 fold in 18-4/EIAV cells treated with the tether; no reductionin infectious virus was observed in Cf2th/EIAV (FIG. 6). These resultsdemonstrate that treatment of lentivirus-infected cells expressing theluciferase gene with a hypericin-luciferin tether generates sufficientchemiluminescence to activate the antiviral activity of hypericin.Subsequent experiments have repeatedly shown that production ofinfectious virus is reduced approximately 90% in 18-4/MA-1 cells ascompared to Cf2th/MA-1 cells.

EXAMPLE 9 Preparation Of Retroviral Vectors and Destruction ofNeoplastic Cells

Construction of Retroviral Vectors for Gene Therapy

The retroviral plasmid vector PLXSN is used for construction of a viralvector for in vitro and in vivo delivery of the luciferase gene.Cell-specific regulation of luciferase expression can be achieved usingspecific promoters to regulate luciferase transcription. For treatmentof HIV infections, a suitable promoter contains the HIV TAR and upstreamSp-1 and NF-kB sites, as shown in FIG. 3B. An example, as shown in FIG.3C, of tumor-specific expression includes the use of the tissue-specifictyrosinase promoter for treatment of primary and metastatic melanoma.See, e.g., Vile et al., Cancer Res., 54, pp. 6228-6234 (1994), Vile andHart, Cancer Res., 53, pp. 3860-3864 (1993), each of which isincorporated herein in its entirety by reference. In all cases,construction of retroviral vectors follows general procedures, asfollows. pLXSN contains NEOr for selection of producer clones and targetcells. NEOr is driven from the SV40 early promoter, and an upstreammultiple cloning site is available for insertion of aluciferase/promoter cassette. In general, pLXSN is linearized andligated to gene cassettes using BamHl. Following transformation,colonies are screened by hybridization using insert-specific probesradiolabelled with ³² P, and positive colonies are characterized byrestriction enzyme digestion and nucleotide sequence analysis.

For production of virus, plasmids are introduced into the packaging cellline PA317 (ATCC CRL 9078) using the calcium phosphate method oftransfection. Two days following infection, cells are subcultured in thepresence of 2 mg/ml G-418, and producer cell clones are isolated usingcloning rings. Each clone is initially tested for production of vectorvirus using a reverse transcriptase assay (Carpenter and Kraus, 1991).Clones shown to produce packaged vector virus are further analyzed fortheir ability to transfer neomycin resistance to NIH/3T3 cells.Supernatant is serially diluted and inoculated in duplicate onto NIH/3T3cells in polybrene (8 μg/ml). Cells are cultured in the presence ofG-418 for 7-10 days, at which time the cells are stained with methyleneblue and the number of neomycin resistant clones is quantitated. Cloneswith a titer greater than 10⁵ are expanded, and DNA is isolated forSouthern analysis of the vector sequences. For production ofsupernatant, producer cells are seeded in 75 cm² flasks and grown until80-90% confluent at which time media is aspirated and replaced with 20ml fresh media. Supernatant is collected at 24 hours intervals for threedays, clarified by centrifugation, aliquotted, and stored at -70° C.

Assays for Antiviral Activity

Supernatants from high producer packaging cell clones are inoculatedonto target cells in 8 μg/ml polybrene. Following transduction, cellsare cultured in the presence of G-418 (Sigma Chemical Co.) in order toselect for clonal populations of transduced cells. Expanded clones areassayed for luciferase expression as described above. Cell clones foundto express high levels of luciferase are infected with virus as beforeand incubated with 30 μg/ml tethered molecule for 30 min in the dark.Following treatment, culture supernatant is sampled daily and assayedfor quantity of infectious virus.

Assays for Tumor Cell Cytotoxicity

Tumor cell cytotoxicity is tested using B16-F1 murine melanoma cells(ATCC CRL 6323) and NIH3T3 (ATCC CRL 1658) fibroblasts as positive andnegative controls, respectively. Supernatants from high producerpackaging cell clones are inoculated onto target cells in 8 μg/mlpolybrene. Following transduction, cells are cultured in the presence ofG-418 in order to select for clonal populations of transduced cells.Expanded clones are assayed for luciferase expression as describedabove. Cell clones found to express high levels of luciferase areincubated with 30 μg/ml tethered molecule for 30 min in the dark, and10-fold serial dilutions are plated in medium containing G-418. After 10days, tumor cell cytotoxicity is evaluated by counting the number ofsurviving cell clones in tether-treated cultures versus non-treatedcultures (Vile and Hart, 1993).

While the invention has been described with reference to specificembodiments, it will be apparent to those skilled in the art that manyalternatives, modifications, and variations may be made. Accordingly, itis intended to embrace all such alternatives, modifications, andvariations that may fall within the spirit and scope of the appendedclaims.

We claim:
 1. An antiviral composition comprising:a. a photosensitizingchemical, virucidally activated by absorbing light or energy within aspecific wavelength range, selected from the group consisting ofhematoporphyrin analogs, polycyclic quinones, phthalocyanines andporphyrins; and b. an energy donating chemical that, when activated,tranfers energy or emits light within the range of that absorbed by saidphotosensitizing chemical.
 2. The composition of claim 1 wherein amolecule of said photosensitizing chemical and a molecule of said energydonating chemical are connected to each other by a chemical tether. 3.The composition of claim 2 wherein said photosensitizing chemical andenergy donating chemical comprise a tethered compound having thefollowing formula: ##STR25## wherein R₁ is said tether, and X and X' aredefined as in claim 7; andwherein R₁ is --CO₂ (CH₂)_(n) Z, wherein n is2 to 15; R₂ -R₄ are H; Z is O, NH or S; A is O or H,H; and G is CH₃, CO₂H, CO₂ Me or CH₂ Br.
 4. The composition of claim 2 wherein saidphotosensitizing chemical and said energy donating chemical comprise atethered compound having the following formula: ##STR26## wherein R₂ issaid tether, and X and X' are defined as in claim 7; andwherein R₂ isselected from the group consisting of --(CH₂)_(n) COZ and --S(CH₂)Z,wherein n is 1 to 15; R₁, R₃, and R₄ are H; Z is O, NH or S; A is O orH, H; and G is CH₃, CO₂ H, CO₂ Me or CH₂ Br.
 5. The composition of claim2 wherein said photosensitizing chemical and said energy donatingchemical comprise a tethered compound having the following formula:##STR27## wherein R₃ is said tether and X and X' are defined as in claim7; andR₃ is selected from the group consisting of --COZ and --(CH₂)_(n)Z, wherein n is 1 to 15; R₁ -R₂ and R₄ are H; Z is O, NH, or S; A is Oor H,H; and G is CH₃, CO₂ H, CO₂ Me or CH₂ Br.
 6. The composition ofclaim 2 wherein said photosensitizing chemical and said energy donatingchemical comprise a tethered compound having the following formula:##STR28## wherein R₄ is said tether and X is defined as in claim 7;andR₄ is --(CH₂)_(n) Z, wherein n is 2 to 15; R₁ -R₂ and R₄ are H; Z isO NH, or S; A is O or H,H, and G is CH₃, CO₂ H, CO₂ Me or CH₂ Br.
 7. Thecomposition of claim 2 wherein said tethered compound has the followingformula: ##STR29## wherein R₁ is H, an alkyl or aryl; R₂ and R₃ are H.OH, Cl, CN or an alkyl; R₄ is H, OH, an alkyl or aryl, R₅ is H or CH₃and R₆ is H, OH, Cl or an alkyl; X₁ is O, N-alkyl, N-aryl or S and X₂ isO, N-alkyl or S; and n is 1 to 10; and m is 1 to
 4. 8. The compositionof claim 1 further comprising a means for activating the transfer ofenergy or the emission of light from said emergy donating chemical. 9.The composition of claim 8 wherein said means for activating saidtransfer or emission comprises an expression plasmid containing a geneencoding luciferase or another activating chemical.
 10. The compositionof claim 9 wherein said gene is under the control of a promoter that istransactivated by replication of a virus.
 11. The composition of claim10 wherein said virus is a lentivirus and wherein said promoter is alentivirus long terminal repeat or a portion thereof.
 12. Thecomposition of claim 1 wherein said photosensitizing chemical is aquinone or porphyrin and said energy donating chemical is luciferin oran analog thereof.
 13. The composition of claim 12 wherein saidphotosensitizing chemical is hypericin or an analog thereof.
 14. Thecomposition of claim 13 wherein said hypericin analog has the followingformula: ##STR30## wherein R₅ is H or CH₃ ; R₆ is H, OH, Cl or an alkyl;and wherein m is 1-4.
 15. The composition of claim 12 wherein saidluciferin analog has the following formula: ##STR31## wherein R₁, R₂, R₃or R₄ is said tether, wherein X and X' are selected from the groupconsisting of S, O, H,H, CH═CH or NR₄ ;if R₁ is said tether, R₁ is --CO₂(CH₂)_(n) Y, wherein n is 2 to 15; R₂ -R₄ are H; and Y is OH, SH, NH₂ ;if R₂ is said tether, R₂ is --(CH₂)_(n) CO₂ H and --S(CH₂)_(n+1) Y,wherein n is 1-15; R₁ and R₃ -R₄ are H; and Y is OH, NH₂ or SH; if R₃ issaid tether, R₃ is selected from the group consisting of --CO₂ H and--(CH₂)_(n) Y, wherein n is 1-15; R₁, R₂ and R₄ are H; and Y is OH, NH₂or SH; and if R₄ is said tether, R₄ is --(CH₂)_(n) Y, wherein n is 2-15;R₁ -R₃ are H; and Y is OH, NH₂ or SH.